Sensor for luminescense-optical determination of an analyte

ABSTRACT

The invention relates to an optochemical sensor functioning in accordance with the FRET-principle and exhibiting an acceptor (chromophore or luminophore) responsive to an analyte contained in a sample medium as well as a donor (luminophore), characterized in that acceptor and donor are located in separate chemical phases, whereby the phase containing the donor is essentially impermeable to the sample medium or to components of the sample medium affecting the luminescence characteristics of the luminophore. The invention further relates to a method for qualitative and/or quantitative determination of at least one analyte and/or component of a gaseous or liquid measuring medium according to the FRET-principle, characterized by the use of the sensor according to the invention.

[0001] The invention relates to a luminescence-optical method forqualitative and quantitative determination of at least one analyteand/or component of a liquid measuring medium containing a chromophore(or a luminophore) which is directly or indirectly responsive to thecomponent to be determined by changing its absorption spectrum, and aluminophore which is not responsive to the component to be determined,where there is at least partial overlap between the emission spectrum ofthe luminophore and the absorption spectrum of the chromophore, andwhere the nonradiative energy transfer between luminophore andchromophore produces a measurable change in at least one luminescencecharacteristic of the luminophore. That principle is known as theso-called FRET-principle.

[0002] The invention further relates to an optochemical sensor forquantitative determination of at least one analyte and/or component of agaseous or liquid measuring medium containing a chromophore (or aluminophore) which is directly or indirectly responsive to the componentto be determined by changing its absorption spectrum, and a luminophorewhich is not responsive to the component to be determined, where thereis at least partial overlap between the emission spectrum of theluminophore and the absorption spectrum of the chromophore, and wherethe nonradiative energy transfer between luminophore and chromophoreproduces a measurable change in at least one luminescence characteristicof the luminophore.

[0003] In the following, luminophores are understood as dyes which emitphosphorescence or fluorescence radiation upon suitable excitation. Theabsorption spectrum of the chromophore is influenced either directly bythe component to be measured or indirectly by a chemical reactionproduct of the component to be measured. The term “quantitativedetermination of a chemical component” refers to the determination ofconcentration and activity as well as gas partial pressure, the valuesof at least one luminescence characteristic of the luminophore beingused to infer the measured quantity.

[0004] A method and a sensor in which pH- and cation-sensitivechromophores (acceptor) are attached, preferably covalently, to aluminophore (donor) are known from U.S. Pat. No. 5,232,858. The pH-valueand/or concentration of the cation to be determined in the measuringmedium is derived from the luminescence decay time of the luminophore.

[0005] As far as the state of the art is concerned, U.S. Pat. No.5,648,269 is also to be mentioned. This document suggests theapplication of the apparent luminescence decay time of the luminophorefor determining the measured quantity. With luminophores with one decaytime component, the apparent luminescence decay time is identical withthe effective decay time. With luminophores with several decay timecomponents, it is easier to evaluate the apparent decay time, however—inparticular with systems that are not robust—there is the drawback ofincreasing errors.

[0006] Luminescence decay times may be obtained by means ofphase-modulation or time-resolved luminescence measuring techniques,respectively.

[0007] A similar method is known from EP-A-0 214 768.Therein, theconcentration of the parameter to be determined in the measuring mediumis inferred from the luminescence intensity measured.

[0008] The rate of nonradiative energy transfer from donor to acceptormolecules depends on the spatial proximity of the molecules of the twosubstances. The transfer rate k_(T)(r) is extremely responsive to thespatial distance r between donor and acceptor and decays with the sixthpower of the distance${{k_{T}(r)} = {\frac{1}{\tau_{D}}\left( \frac{R_{o}}{r} \right)^{6}}},$

[0009] whereby τ_(D) indicates the luminescence decay time of the donorin absence of the acceptor and Ro indicates the characteristic Försterdistance. The latter is that donor-acceptor distance in which a 50%efficiency of the energy transfer is provided. Depending on therespective donor-acceptor pair, Ro is between 2 and 9 nm.

[0010] Due to the nonradiative energy transfer from donor to acceptormolecules, the macroscopically determinable values of theluminescence-optical parameters (luminescence quantum efficiency,luminescence intensity, luminescence decay time) of the luminophore willundergo a particularly efficient change if a substantial number ofmolecules of the two substances are brought into close spatial contactwith each other.

[0011] To obtain close spatial contact, U.S. Pat. No. 5,232,858 proposesa covalent bonding of donor and acceptor molecules. In EP-A-0 214 768individual donor and acceptor molecules are covalently attached to thesurface of a joint substrate, such as glass.

[0012] The covalent bonding of donor and acceptor molecules as describedin U.S. Pat. No. 5,232,858 has the advantage that the mean spatialdistance of donor/acceptor may be kept as constant as possible. However,it is a disadvantage that particularly great synthesis efforts arerequired to produce covalent bonds between desirable luminophores andsuitable pH- or ion-sensitive chromophores.

[0013] Considerable efforts are also needed to covalently attach donorand acceptor molecules to the surface of a joint substrate (EP-A-0 214768), which, above all, brings about the drawback that boundary surfacephenomena impair the quality of the measured results.

[0014] Thus, in U.S. Pat. No. 5,942,189 and U.S. Pat. No. 6,046,055 itis suggested that the luminophore and the chromphore are ionicsubstances of differing electrical charges, which are present as ionicpairs in a matrix material that is permeable to the chemical componentto be determined.

[0015] The use of long-lived luminophores (having luminescence decaytimes >100 ns, preferably >1 μs), such as exhibited, for example, bymetal-ligand complexes, certain porphyrins and lanthanides, is of utmostimportance to a general commercial application. Long-lived luminescenceprovided, the opto-electronic arrangements and components for thedetermination of the luminescance decay time and/or values to be derivedtherefrom (for example, mean luminescance decay time, phase angle) maybe determined in a particularly inexpensive manner by means of phase- ortime-resolved methods.

[0016] However, the inventor of the present application has recognizedthat the above-mentioned, previously known methods bring about themutual disadvantage that in particular the luminescence of long-livedluminophores is influenced by a number of components of the measuringmedium. A known characteristic of such luminophores is the particularlygreat dependency of the luminescence characteristic on the O2 content ofthe sample. Consequently, such luminophores are thus often used fordetermining the O2 content (EP-A-0 907 074). When using thoseluminophores as donor dyes with sensors based upon the FRET-principle,it is thus necessary to exactly know or determine the O2 content of themeasuring medium and to carry out appropriate adjustments. Examples offurther substances having an influence on the luminescence quantumefficiency are amines and water. In the course of continuousmeasurements (monitoring), luminescence dyes may be completely orpartially destroyed by the emerging singlet-O2. Accordingly, additivesfor limiting that process were suggested. However, a general drawback ofknown, advantageous luminescence dyes is the luminescencecharacteristics' sensitivity to minor changes of the chemical-physicalmicroenvironment, caused by any components of the sample, in particularwater. In case of sample media of unknown and/or variable chemical orbiochemical compositions, that leads to a significant limitation of themeasuring accuracy. For example, in medical diagnostics,reproducibilities of +/−5 milli-pH are expected in the field of blood-pHdetermination.

[0017] It is the object of the invention to improve luminescence-opticaldetermination methods based upon the FRET-principle, which are basedupon a luminophore (donor) and a chromphore (acceptor, indicator)reversibly binding the substance (analyte) to be determined or itsreaction products, with regard to the susceptance to failure caused bycomponents of the sample to be measured. Furthermore, particularly greatchemical synthesis steps needed to obtain the spatial proximity of asubstantial number of acceptor molecules and shielded donor moleculesare to be avoided.

[0018] According to the invention, this object is achieved in that theluminophore (donor) and the chromophore (acceptor) are located indifferent chemical phases. The matrix material of the donor phase is tobe essentially impermeable to components of the sample medium affectingthe luminescence characteristics of the donor. The matrix material ofthe acceptor phase is to be permeable to the analyte or its reactionproducts, respectively.

[0019] Thus, the invention relates to an optochemical sensor functioningin accordance with the FRET-principle and containing a chromophore aswell as a luminophore responsive to an analyte contained in a samplemedium and is characterized in that the luminophore and the chromophoreare located in separate chemical phases, whereby the phase containingthe luminophore is impermeable to the sample medium or to components ofthe sample medium affecting the luminescence characteristics of theluminophore.

[0020] It is provided in a variant of the sensor according to theinvention that the matrix material of the acceptor and donor phases isprovided in mixed form or in the form of a thin layer (sensor layer),which may be attached to a transparent substrate or to a light guide. Todecouple the sensor layer from possible disturbing optical interactionswith the substance to be analyzed, an optical insulation layer permeableto the analyte and located between the sensor layer and the substance tobe analyzed is optionally provided.

[0021] In a further embodiment (FIG. 5), the sensor layer consists of athin porous material. The pores of that material contain the matrixmaterial of the donor phase and the matrix material of the acceptorphase.

[0022] In a further embodiment (FIG. 6), the sensor layer is composed ofa thin film representing the matrix material of the acceptor phase.Thereby, the matrix material of the donor phase is provided inhomogeneously distributed fashion in the form of particles in the matrixmaterial of the acceptor phase. The acceptor phase contains the acceptormolecules being homogeneously distributed and provided in a sufficientconcentration so that an adequate number of acceptor molecules arelocated in the spatial proximity to the donor molecules provided in thedonor phase which is necessary for the nonradiative energy transfer.

[0023] In a further embodiment (FIG. 7), the acceptor molecules arebound to the surface of the particles. Thereby, the chromophore may bebound at the surface adsoptively or electrostatically or, mostpreferably, covalently to functional groups. The bonding of thechromophore to the particle surface above all has the advantage that a)less chromphores are necessary, b) optical filter effects caused byexcessive chromophore concentrations of the acceptor phase may beavoided, and c) the ratio of donor and acceptor molecules alsodetermining the rate of energy transfer may be adjusted already on thelevel of producing the particles, thus being independent of theconcentration of the acceptor molecules in the acceptor phase.

[0024] The method according to the invention serves for the qualitativeand/or quantitative determination of at least one analyte and/orcomponent of a gaseous or liquid measuring medium in accordance with theFRET-principle and is characterized by the use of a sensor according tothe invention.

[0025] Preferably, the method according to the invention serves for thedetermination of the pH-value of a sample, for the determination ofconcentrations and/or activities of ions in a sample or for thedetermination of components exhibiting acid or alkaline reactions in anaqueous medium while being gaseous under normal conditions.

[0026] In particular, the method according to the invention serves forthe determination of concentrations and/or activities of Li⁺, Na⁺, K⁺,Mg⁺⁺, Ca⁺⁺ or Cl⁻.

[0027] Furthermore, the method according to the invention preferablyserves for the determination of CO₂ or NH₃ in a liquid measuring medium.

[0028] Further areas of application of the method according to theinvention concern so-called transducers. Therein, the chromophore doesnot directly react with the analyte, but indirectly. Examples of thatare so-called enzymatical sensors (for example, for determining urea andcreatinine). Thereby, one or more enzymes react with the analyte,leading to the formation of a product which reacts directly with thechromophore.

[0029] Preferably, the measuring medium is a body fluid, in particularblood, plasma or serum.

[0030] Most advantageous luminophores (donor) used according to theinvention are those which feature high luminescence quantum efficiencyand long luminescence decay time (>100 ns). Preferred luminophores arecationic, metalorganic complexes of palladium, rhodium, platinum,ruthenium, osmium, rare earths (in particular, europium and lanthanum).The organic portion of these metalorganic complexes may consist, forexample, of ligands from the group of porphyrins, bipyridyls,phenanthrolines or other heterocyclical compounds.

[0031] Preferred pH- and cation-sensitive chromophores (acceptor) areanionic substances whose light absorption will change upon direct orindirect chemical/physical interaction with the component of the samplemedium to be determined, and whose absorption spectrum overlaps theemission spectrum of the luminophore, at least partially.

[0032] I) Determination of the pH-Value of a Sample

[0033] Optical sensors for pH determination according to the state ofthe art (cf M. J. P. Leiner and O. S. Wolfbeis “Fiber Optic pH Sensors”in O. S. Wolfbeis “Fiber Optic Chemical Sensors and Biosensors”,CRC-Press, Boca Raton, 1991, Vol. I, Chapter 8) usually contain anabsorption dye (chromophore) or fluorescent dye incorporated in anion-permeable, preferably hydrophilic polymer matrix. In dependence ofthe pH-value (pH=−log(aH+)) of the sample medium, a thermodynamicequilibrium is established between the protonated and deprotonated formsof the chromophore or fluorophore, respectively. From the concentrationratio of the two forms measurable by optical methods, the pH-value ofthe sample medium may be obtained.

Equilibrium $K_{d} = \frac{{cA}^{-}*{cH}^{+}}{cAH}$

[0034] AH is the protonated, and A⁻ is the deprotonated form of thepH-sensitive Chromophore. H⁺ denotes a proton. K_(d) is the equilibriumconstant. c denotes the concentration.

[0035] In U.S. Pat. No. 5,232,858 initially mentioned, pH-sensitivechromophores are described, which are attached, preferably covalently,to a pH-insensitive luminophore (donor). From the luminescence decaytime of the luminophore (L), the pH-value of the test solution isobtained.

[0036] For luminescence-optical pH-determination according to theinvention, for example, a pH-sensitive chromophore is used as anacceptor dye, which is provided in the acceptor phase or is at least indirect contact with the acceptor phase, respectively, whereby theacceptor phase may also be the medium to be analyzed (measuring medium).

[0037] In the instance of low pH-values (pH<<pK of the chromophore) ofthe sample medium, the chromophore is present in fully protonated form.Due to the minimal spectral overlap of the absorption band of thedeprotonated chromophore and the emission band of the luminophore, thenonradiative energy transfer rate from luminophore to chromophorereaches a minimum. Correspondingly, the values of mean luminescencedecay time and relative luminescence intensity of the luminophore reacha maximum.

[0038] In the instance of high pH-values (pH>>pKa of the chromophore) ofthe sample medium, the chromophore is present in fully deprotonatedform. Due to the maximal spectral overlap of the absorption band of thedeprotonated chromophore and the emission band of the luminophore, thenonradiative energy transfer rate from luminophore (donor, donor dye) tochromophore (acceptor, acceptor dye) reaches a maximum. Correspondingly,the values of mean luminescence decay time and relative luminescenceintensity of the luminophore reach a minimum.

[0039] For pH-values of the sample medium in the range of +/−1.5 pHunits of the pKd value (pKd=−log Kd) of the chromophore, the pH-value ofthe sample medium may be inferred with sufficient accuracy from the meanluminescence decay time or relative luminescence intensity of theluminophore.

[0040] II) Determination of Concentrations and/or Activities of Cationsand Anions in a Sample (Li+, Na+, K+, Mg++, Ca++, Cl−)

[0041] Previously known optical sensors and optical measuring methods,respectively, for determining the concentrations and/or activities ofcations in a sample medium are based upon different methods. U.S. Pat.No. 5,232,858 as initially mentioned describes cation-sensitivechromphores which are attached, preferably covalently, to acation-insensitive luminophore.

[0042] In the instance of very high cation concentrations (cY^(+p)>>Kdof the chromophore) of the sample medium, the chromophore is present infully complexed form. (Y is the cation to be determined, +p is itsatomic number.) In the instance of very low cation concentrations(cY^(+p)<<Kd of the chromophore) of the sample medium, the chromophoreis present in free, noncomplexed form.

[0043] If the logarithmic concentration log(cY^(+p)) of the cation to bedetermined of the sample medium is in the range of −log(Kd)+/−1.5, theconcentration of the cation to be determined in the sample medium may beinferred with sufficient accuracy from the mean luminescence decay timeand/or relative luminescence intensity of the luminophore.

[0044] Further optical measuring methods and sensors, respectively, fordetermining the concentrations and/or activities of cations are known,for example, from U.S. Pat. No. 4,645,744, EP-A-0 358 991 and EP-A-0 461392, where a pH-sensitive chromophore or a pH-sensitive luminophore,respectively, and a neutral ionophore are provided in a substantiallyhydrophobic polymer matrix. The disclosed measuring method is based onthat cations (Y^(+p)) are exchanged with the sample medium (for example,K⁺ for H⁺ or Ca⁺⁺ for 2H⁺). As a consequence, the measured results aredependent on the pH-value of the sample medium. Such measuring methodsare suitable under measuring conditions in which the pH-value of thesample medium is known or may be adjusted to a known value by means of apH buffering layer.

[0045] In further development according to the invention of thesemethods, a pH-sensitive chromophore located in the acceptor phase or atleast contacting the same and a pH-insensitive luminophore located inthe donor phase as well as a neutral ionophore (I) selective for thecation to be determined and located in the acceptor phase are providedfor determination of the cationic concentration.

[0046] From EP-A-0 358 991 and Anal.Chim.Acta 255(1991), p.35-44,optical sensors for determining anions, for example Cl⁻, are known,whereby the anion to be determined is co-extracted from the measuringmedium together with a cation (for example, Cl⁻ and H⁺). In thatinstance, a lipophilic, pH-sensitive chromophore (fluoresceinderivative) and an optically inactive, cationic substance (Q⁺) areprovided in a substantially hydrophobic polymer matrix.

[0047] In dependence of the H⁺ and Cl⁻ concentrations of the measuringmedium, the pH-sensitive chromophore located in the polymer matrix ispresent in protonated and/or deprotonated form. Absorption of thedeprotonated form will rise with a growing degree of deprotonation. Thedegree of deprotonation (and hence, absorption) depends on the pH-valueand concentration of the anion to be determined. The pH-value of themeasuring medium must be known or adjusted to a known value, so as toindicate the concentration of the anion to be determined.

[0048] In further development according to the invention of thedisclosed method, for example, a pH-sensitive chromophore located in anessentially hydrophobic acceptor phase permeable to chloride ions of themeasuring medium by means of co-extraction or contacting the same(whereby the acceptor phase CANNOT be the measuring medium), aluminophore located in a donor phase as well as a lipophilic, cationicsubstance (ionophore) are provided for determination of the chlorideconcentration of a measuring medium. The lipophilic substance (Q+) maybe a quaternated ammonium compound, for instance.

[0049] Examples of pH-sensitive chromophores used according to theinvention are listed below in Table 1. In the instance of low pH-valuesand high chloride concentrations of the measuring medium, thepH-sensitive chromophore preferably is present in protonated form andthe optically inactive, cationic substance forms a counterion to Cl⁻ inthe matrix. Absorption of the deprotonated form of the chromophorereaches a minimum. The values of mean luminescence decay time andrelative luminescence intensity of the luminophore reach a maximum.

[0050] In the instance of high pH-values and low chloride concentrationsof the measuring medium, the pH-sensitive chromophore preferably ispresent in deprotonated form, the optically inactive, cationic substancecompensating the negative charge generated by dissociation of theproton. Absorption of the deprotonated form of the chromophore reaches amaximum. The values of mean luminescence decay time and relativeluminescence intensity of the luminophore reach a minimum.

[0051] If the pH-value of the measuring medium is known, the chlorideconcentration of the measuring medium may be inferred from the values ofmean luminescence decay time and/or relative luminescence intensity ofthe luminophore.

[0052] III) Determination of Components of Liquid or Gaseous MeasuringMedia, which Exhibit Weak Acid or Basic Reactions in AqueousEnvironments and are Gaseous under Normal Conditions:

[0053] Determination of CO₂

[0054] Optical sensors for determination of the CO₂ partial pressure ofa liquid or gaseous measuring medium usually comprise a reaction spacewhich is separated from the medium being measured by an ion-impermeable,gas-permeable material. The reaction space is often identical with theindicator substrate material of an optical pH sensor. In addition, thereaction space usually includes one or several pH buffering substances,such as carbonates, phosphates, and/or organic compounds exhibiting acidor basic reactions in aqueous media. As a consequence, the pCO₂determination of the measuring medium may be traced back to optical pHdetermination.

[0055] In a variant of this measuring principle described in EP-A-0 105870, the reaction space is provided in the shape of “droplets” in anion-impermeable, gas-permeable polymer material. In a variant of thismeasuring principle described in U.S. Pat. No. 5,496,521, the reactionspace is provided in the shape of a hydrophilic layer covered by anion-impermeable, gas-permeable polymer material.

[0056] AH is the protonated, and A⁻ is the deprotonated form of thechromophore. H⁺ denotes a proton.

[0057] In further development according to the invention of theluminescence-optical CO₂ determination, a donor shielded from chemicalparameters according to the invention and a pH-sensitive acceptor dyeare provided in a hydrophilic reaction space separated from themeasuring medium by an ion-impermeable, gas-permeable material.

[0058] CO₂ determination by means of the sensors according to theinvention comprising a reaction space containing an aqueous pH bufferand an ion-impermeable, gas-permeable material separating the reactionspace from the measuring medium is traced back to the determination ofthe pH-value in the reaction space of the sensor. High CO₂ values of themeasuring medium correspond to low pH-values of the reaction space, andlow CO₂ values of the measuring medium correspond to high pH-values ofthe reaction space.

[0059] An alternative method of optical CO₂ determination, for whichonly a single reaction space without the aid of aqueous pH bufferingsubstances is required, is described by Mills et al. in Anal. Chem. 64,1992, 1383-1389.In that case, the deprotonated form of a pH-sensitivechromophore with an optically inactive, cationic substance (Q⁺) ispresent in a reaction space consisting of an essentiallyion-impermeable, gas-permeable polymer material. CO₂ of the measuringmedium diffusing into the polymer material is hydrated and reacts in achemcial equilibrium reaction with the deprotonated form of thechromophore and the optically inactive substance. From the lightabsorption of the deprotonated form of the chromophore, the CO₂ partialpressure of the measuring medium is inferred.

[0060] In a variant according to the invention of theluminescence-optical CO₂ determination, an essentially lipophilic,pH-sensitive chromophore located in an acceptor phase essentiallyimpermeable to ionic substances in the instance of aqueous measuringmedia or at least contacting the same, a luminophore located in a donorphase as well as an essentially lipophilic, cationic substance locatedin the acceptor phase are provided.

[0061] According to the invention, CO₂ determination by means of asensor comprising a reaction space without aqueous buffering substancesis traced back to the determination of the ratio of the protonated anddeprotonated forms of a pH-sensitive chromophore.

[0062] In the instance of very low CO₂ values of the measuring medium,the chromophore preferably is present in deprotonated form and forms aionic bond with the optically inactive, cationic substance in thematrix. Absorption of the deprotonated form of the chromophore reaches amaximum. The values of mean luminescence decay time and relativeluminescence intensity of the luminophore reach a minimum.

[0063] In the instance of high CO₂ values of the measuring medium, thechromophore is present in protonated form. The optically inactive,cationic substance forms a ionic bond with hydrogen carbonate.Absorption of the deprotonated form of the chromophore reaches aminimum. The values of mean luminescence decay time and relativeluminescence intensity of the luminophore reach a maximum.

[0064] Determination of NH₃

[0065] The determination of NH₃, as an example of a component exhibitinga basic reaction in aqueous environment, may be effected in a waysimilar to the determination of CO₂ according to Mills. (T. Werner etal., Analyst 120, 1995, 1627-1631). No optically inactive, cationicsubstance is necessary.

[0066] The determination of NH₃ according to the invention is done byanalogy with the CO₂ determination according to the invention:

[0067] pH-sensitive chromophores suitable for use according to theinvention: TABLE 1 pH-sensitive chromophores Absorption wavelength [nm]Chromophore protonated/deprotonated pKa Triphenylmethane dyes:Bromophenol blue 430/617 3.8 Bromothymol blue 430-435/615-618 6.7Dibromoxylenol blue 420/614 7.6 Azo dyes: Calmagit 530/605 8.0 Nitrazineyellow 460/590 6.5 Others: o-chlorophenol-indophenol 555/625 7.1Naphthol-phthalein 428/661 6.7, 7.9

[0068] In addition, pH-sensitive triphenylmethane dyes of the generalform

[0069] are used. Independently of each other, R1-6 may be H, halogenatoms, nitro groups and alkyl groups. X⁻ stands for an optional groupfor covalently immobilizing the chromophore. Suitable groups are, forexample —(CH2)n-SO3⁻ or —(CH2)n-COO⁻, —(CH2)n-NH2 (n=0-18).

[0070] pH-sensitive azo dyes of the general form

[0071] wherein, independently of each other, R1-R4 stand forsubstituents, such as halogen atoms, nitro groups or alkyl groups,respectively, and groups suitable for covalent immobilization, whereby,however, at least one —OH group has to be present.

[0072] Cation-sensitive chromophores (chromoionophores) suitable for useaccording to the invention:

[0073] Examples of cation-sensitive chromoionophores usable according tothe invention for determination of lithium, potassium, sodium, magnesiumand calcium ions include anionic azo dyes, stilbene dyes andmerocyanines, which contain at least one ion-selective group (ionophoregroup) and whose absorption band with the longest wavelengths overlapsthe emission band of the luminophore at least partially, the interactionwith the cations to be determined leading to a spectral shift of theabsorption band with the longest wavelengths.

[0074] Neutral ionophores are listed below in Table 2. TABLE 2 Neutralionophores (suitable for ion exchange) Sensors Ionophor Ion PTM14C4(14-crown-4)**) Li+ Sodium Ionophore I-II*) Na+ Valinomycin K+ MagnesiumIonophore ETH 3832*) Mg++ Calcium Ionophore I-IV*) Ca++

[0075] TABLE 3 Luminophore (donor dyes) Absorption Luminescence maximummaximum Luminophore (L) Abbrev. (nm) (nm) (Ru(II)-tris-2,2′- Ru(bpy)₃ ²⁺452 628 bipyridyl)²⁺ (Ru(II)-tris-2,2′ 4,4- Ru(dph-bpy)₃ ²⁺ 474 632diphenyl bipyridyl)²⁺ (Ru(II)-tris-1,10- Ru(phen)₃ ²⁺ 447 604phenanthroline)²⁺ (Os(II)-bis-terpyridine)²⁺ 510 729 (Os(II)-tr-1,10-650 690 phenanthroline)²⁺

[0076] Essentially hydrophobic, ion-impermeable acceptor phases:

[0077] To produce sensors according to the invention, which exchange thecation to be determined (such as K+) with a proton of the measuringmedium or co-extract the anion to be measured (such as Cl−) with aproton of the measuring medium, polymer materials substaniallyimpermeable to ionic substances of the measuring medium are suitable.

[0078] These materials are also suitable for preparing sensors accordingto the invention which are used for determination of gases and/orgaseous components in liquid measuring media.

[0079] Preferred are all essentially hydrophobic polymers that aresoluble in organic solvents, such as polyvinyl chloride, polystyrenes,polycarbonates, polyethylenes, polyurethanes, silicones, copolymers ofpolyvinyl alcohol and polyvinyl acetate, and copolymers of polyvinylchloride, polyvinyl alcohol and/or polyvinyl acetate.

[0080] Up to 80% by weight plasticizers may be added to these materials,such as dioctyl sebacate, tris-(2-ethylhexyl)-phosphate,2-nitrophenyl-octyl-ether, 2-nitrophenyl-butyl-ether.

[0081] Essentially hydrophilic, ion-permeable acceptor phases:

[0082] To produce sensors according to the invention with pH andion-sensitive chromophores, hydrophilic, ion-permeable polymers arepreferred.

[0083] Examples of that are cellulose, polyurethanes with hydrophilicgroups, polyhydroxyethylmethacrylates, crosslinked polyvinyl alcoholsand/or polyacrylamides.

[0084] Materials suitable for donor phases include, for example, hard,unplasticized polymers such as polyacryl nitrile plus derivatives, PVCand polyvinylidene chloride.

[0085] The invention will be explained in greater detail with referenceto FIGS. 1-7, wherein

[0086]FIG. 1 displays a calibration curve of a pH reagent;

[0087]FIG. 2 displays a calibration curve of a pH sensor according tothe invention;

[0088]FIG. 3 displays a calibration curve of a CO₂ sensor according tothe state of the art;

[0089]FIG. 4 displays a calibration curve of a CO₂ sensor according tothe invention and

[0090]FIG. 5 to 7 each show an embodiment of a sensor according to theinvention in schematic representation, such as described on page 5.

[0091] Determination of calibration values:

[0092] The determination of the calibration curves (FIGS. 1-4) and theO2 dependencies was carried out by determining the phase shift of theluminescence with regard to the excitation light modulated in sinusoidalfashion. Due to the use of a long-lived luminophore, a simple measuringarrangement exhibiting a modulation frequency of 45 kHz is sufficient.

[0093] For the purpose of measuring, sensor disks were punched from thesensor foils, they were attached to the end piece of a two-armed lightguide bundle and were contacted with the respective measuring media, orthe end piece of the light guide bundle was dipped directly into themeasuring medium containing the sample. A blue LED (470 nm) suppliedwith an amplitude voltage of 5V, which had been modulated up to 45 kHzin sinusoidal fashion, was used as an excitation light source. Blue foilfilters were used as filters for the excitation light. The excitationlight was guided to the sensor foil or to the test liquid, respectively,by means of light guides. The emitted luminescence light was guided to afilter, a combination of a OG 570 glass filter (Schott) and a red foilfilter, by means of a light guide bundle and was further guided onto adetector (photo multiplier, type Hamamatsu H5702). The distributionvoltage modulated in sinusoidal fashion of the LED and the signal of thedetector were evaluated by means of a lock-in amplifier. The phase shiftφ was obtained as a measuring signal. The decay time T was calculatedfrom the measured phase shift φ and the modulation frequency f=45 kHzaccording to the following formula: τ=tan(φ)/(2πf).

[0094] Phosphate buffers which had been adjusted to the desiredpH-values with the aid of NaOH or HCl, respectively, were used asmeasuring and/or calibration media for the pH sample (FIG. 1) or the pHsensors (FIG. 2), respectively. The buffers were adjusted to theatmospheric oxygen value by employing tonometrics with air or wererendered oxygen-free by adding Na₂SO₃. Mixed gases of variouscompositions of N₂, O₂ and CO₂ were used as calibration media for CO₂sensors (FIGS. 3 and 4).

[0095]FIG. 1 shows the calibration curve of an optical pH reagent basedupon the FRET-principle (produced in accordance with 2.3) and containinga pH-sensitive acceptor dye, the donor dye being present “in protectedfashion” in a donor phase of the invention. The two curves show thephase angle (ordinate) of the luminescence light of the pH sampledispersed in calibration liquids exhibiting differing pH-values(abscisse).The curve denoted by “O” was taken up when being saturated byair (21.95% O₂). The curve denoted by “N” was taken up when beingsaturated by N₂.

[0096]FIG. 2 shows the calibration curve of an optical pH sensor basedupon the FRET-principle (produced in accordance with 2.4), the donor dyebeing present “in protected fashion” in a donor phase of the invention.The two curves “N” and “O” refer to the same samples as explained forFIG. 1 and depict the phase angle (ordinate) with calibration liquidsexhibiting differing pH-values (abscisse).

[0097]FIG. 3 shows the calibration curve of an optical CO₂ sensor basedupon the FRET-principle (produced in accordance with 3.1), the donor dyebeing present “in unprotected fashion” in the acceptor phase. The twocurves depict the luminescence decay times (ordinate) with calibrationgases of differing partial pressures of CO₂ (abscisse). The curve “OF”means oxygen-free. The curve “AC” means “state of the air” with 21.95%O₂.

[0098]FIG. 4 shows the calibration curve of an optical CO₂ sensor basedupon the FRET-principle (produced in accordance with 3.2), the donor dyebeing present “in protected fashion” in a donor phase of the invention.The two curves (meanings of “OF” and “AC” as in FIG. 3) depict theluminescence decay times (ordinate) with calibration gases of differingpartial pressures of CO₂ (abscisse).

[0099]FIG. 5 shows a preferred embodiment of the sensor according to theinvention. Therein,

[0100] 1 means optically transparent carrier

[0101] 2 means optical insulation layer (optional)

[0102] 3 means light descending on the sensor or emerging therefrom

[0103] 51 means porous material

[0104] 52 means donor phase with donor dye (small squares)

[0105] 53 means acceptor phase with acceptor dye (small circles)

[0106]FIG. 6 shows a further preferred embodiment of the sensoraccording to the invention, with reference numerals 1, 2 and 3 havingthe same meanings as in FIG. 5. Futhermore,

[0107] 61 means donor phase with donor dye (larger circles)

[0108] 62 means acceptor phase with acceptor dye (smaller circles)

[0109]FIG. 7 shows a further preferred embodiment of the sensoraccording to the invention, with reference numerals 1, 2 and 3 havingthe same meanings as in FIG. 5. Futhermore,

[0110] 71 means donor phase with donor dye (larger circles) and withbound acceptor dye (smaller circles)

[0111] 72 means acceptor phase

EXAMPLES Ru(diphphen)₃TMS₂

[0112][Ru(II)-tris-(4,7-diphenyl-1,10-phenanthroline)(3-trimethylsilyl)-1-propanesulphonate] (I. Klimant and O. S. Wolfbeis, Anal.Chem. 67(1995) 3160).

Example 1

[0113] This example shows that a donor dye which, according to theinvention, is embedded in a donor phase exhibits less O2 sensitivitythan the same dye being embedded in the acceptor phase.

[0114] General Description of the Preparation of Nanoparticles withEmbedded Donor Dye

[0115] 1.1 Embedding of a donor dye into an acceptor phase

[0116] 1 mg donor dye Ru(diphphen)₃TMS₂ and 100 mg of the hydrophilicpolymer D4 (polyurethane with hydrophilic sequences; Tyndale PlainsHunter LTD, Ringoes, N.J. 08551, USA) were dissolved in ethanol:water(90:10 w/w). The solution was drawn up a polyester foil (Mylar, Dupont)by knife application. After evaporation of the solvent, a film with alayer thickness of approximately 20 μm emerged. Measuring arrangement(45 kHz, blue LED, OG 570) Dry: air 55.4° N2: 58.3° Water: Airsaturated: 53.5° N2 saturated: 58.4°

[0117] The measuring result shows that the donor dye Ru(diphphen)₃TMS₂being present “in unprotected fashion” in an acceptor phase exhibits anO2 sensitivity of 2.9° (dry acceptor phase) or of 4.9° (wet acceptorphase), respectively, when getting into contact with O2-free (N2saturation) or 21.95% O2 (air) saturated, gaseous and aqueous media.

[0118] 1.2 Preparation of Nanoparticles with Donor Dye

[0119] 400 mg polyacryl nitrile (Polyscience) and 8 mg Ru(diphphen)₃TMS₂were dissolved in 80 ml DMF (Merck). After slowly dripping in 500 ml ofwater, the emerged suspension was mixed with NaCl and was centrifuged.The centrifuge effluent was washed with water several times andsubsequently with ethanol.

[0120] 1.3 Embedding of the Donor Dye Carrying Nanoparticles into aStratified Acceptor Phase

[0121] The ethanolic suspension (of 1.2) was mixed with a solution of400 mg hydrophilic polymer D4 (Tyndale Plains Hunter LTD, Ringoes, N.J.08551, USA) in 5 ml ethanol:water (90:10 w/w). The suspension was drawnup a polyester foil (Mylar, Dupont) by knife application. Afterevaporation of the solvent, a film with a layer thickness ofapproximately 20 μm emerged. Measuring arrangement (45 kHz, blue LED, OG570) Dry: air 57.4°; N2: 58.2° Water: Air saturated: 56.8° O2 free (SO₃²⁻): 58.6°

[0122] The measuring result shows that the donor dye Ru(diphphen)₃TMS₂embedded in a nanoparticle of the invention and thus being present “inprotected fashion” in the donor phase exhibits an O2 sensitivity of0.80° (dry acceptor phase) or of 1.60° (wet acceptor phase),respectively, when getting into contact with O2-free or 21.95% O2saturated, gaseous or aqueous media.

[0123] By comparing these values with the values of 1.1, it thus becomesapparent that one and the same donor dye which is present in a donorphase according to the invention exhibits less O2 sensitivity than thedonor dye which, according to the state of the art, is present directlyin the acceptor phase.

Example 2 (pH Sensor)

[0124] General Description of the Preparation of pH FunctionalNanoparticles and a pH-Sensitive Layer

[0125] In a first step, a OH functional copolymer made up ofacrylonitrile and an OH functional methacrylate is prepared. In a secondstep, nanoparticles containing embedded donor dye are produced from thatpolymer. In a third step, a pH-sensitive acceptor dye is covalentlyattached to the functional groups of the copolymer. In doing so, theacceptor dye is located predominantly in the “soft” hydrophilic regions(acceptor phase) of the particles, thus being accesible for ions. Thedonor dye is dissolved predominantly in the “hard” regions (donorphase), thus making it difficult for interfering substances to gainaccess. In a fourth step, the particles are suspended in a solution of ahydrophilic polymer, and the solution is deposited on a suitable carriermaterial. After evaporation of the solvent, a pH-sensitive sensor filmemerges.

[0126] 2.1 Preparation of the Copolymer

[0127] 230 g de-ionized H₂O were rendered oxygen-free by 2 h of rinsingwith nitrogen. Under stirring and a nitrogen environment, 4 g SDS(sodium dodecyl sulphate p.A., Merck) were dissolved. 20 mlacrylonitrile (Fluka) and 1 ml polyethylene glycolmonometacrylate(Polyscience, n=200, order no.16712) were added to that solution. Thatmixture was taken to 50° and mixed with 4 ml 1 N HCl (Merck).Polymerization was started by adding 400 mg ammonium peroxodisulphate(Merck) and was carried out for 12 h at 50°. The polymer was sucked offand washed several times with water and ethanol. In the following, thispolymer is called PAN-PEG.

[0128] 2.2 Preparation of Nanoparticles with Donor Dye

[0129] 400 mg PAN-PEG and 20 mg of the donor dye Ru(diphphen)₃TMS₂ weredissolved in 80 ml DMF (Merck). After slowly dripping in 500 ml ofwater, the suspension was mixed with NaCl and was centrifuged. Thecentrifuge effluent was washed with water several times.

[0130] 2.3 Binding of the pH-Sensitive Acceptor Dye of the Chromophoreto the Nanoparticles

[0131] 25 mg of the pH-sensitive acceptor dye N9 (Merck) were pulverizedwith 8 Tr. H₂SO₄ conc. (Merck) and were activated for 30 min in a waterjet vacuum. The dye was absorbed in 100 ml de-ionized water and wastaken to pH 7 with the aid of NaOH. The above described centrifugeeffluent was added to this mixture, after 5 min 4.2 g NaCO₃, and after 5min 2 ml 5 M NaOH were added. After further 20 min, acidification to pH3 by means of HCL conc. was carried out. The particles were split off bycentrifugation and were washed with basic buffer, water and ethanol.

[0132] The thus produced particles were suspensed into an aqueousmeasuring medium, and the measurements were carried out at variouspH-values and unter air and N2 saturation. The result is depicted inFIG. 1 and shows that, for the purpose of pH determination, thepH-sensitive particles may be added to the measuring medium also asreagents (=“probe”).

[0133] Measuring medium: pH 7.3 Air saturated: 50.0° O2-free (N2saturated): 51.3°

[0134]2.4 Preparation of the pH-Sensitive Layer (with Particles of 1.3)of an Optical pH Sensor

[0135] The ethanolic suspension (2.3) was mixed with a solution of 400mg hydrophilic polymer D4 (Tyndale Plains Hunter LTD, Ringoes, N.J.08551, USA) in 8 ml 90% (w/w) ethanol. The suspension was drawn up apolyester foil (Mylar, Dupont) by knife application. After evaporationof the solvent, a pH-sensitive film with a layer thickness ofapproximately 20 μm emerged.

[0136]FIG. 2 shows the pH dependency of the phase signal of the thusproduced optical sensor.

[0137] pH 7.5 buffer: Air saturated: 49.3° O2-free (SO₃ ²⁻): 51.30°

[0138] Comparing these measuring results with the data of 1.1 shows thatan optical pH sensor with a donor dye which is present “in protectedfashion” in a donor phase according to the invention exhibits a lower O2sensitivity (change of the phase angle 2.0° at transition O2-free afterair saturation) than the donor dye which is present in the acceptorphase according to the state of the art.

Example 3 (CO₂ Sensor)

[0139] With that sensor, the luminescent nanoparticles are embedded in apolymer film in which the indirectly CO2 sensitive absorption indicatoris present in dissolved form. The difference from the pH sensor (2.4)essentially consists in that, in this case, no covalent coupling of theanalyte-sensitive absorption dye to the nanoparticles takes place.

[0140] 3.1 Embedding of a Donor Dye Used According to the Invention inan Acceptor Phase

[0141] 5 g ethylcellulose (ethoxyl content 46%, Aldrich, Steinheim,Germany) were dissolved in 100 ml of a toluol:ethanol mixture (20:80,v:v). 100 μL tetraoctyl ammonium hydroxide (611 mmol/kg ethylcellulose)and 1 mg m-cresol purple-(24 mmol/kg ethylcellulose) (m-cresolpurple=tridodecyl methylamimonium salt, TDMA) were added to 1 g of thissolution. 0.7 mg of the donor dyeRu(diph-bpy)₃TMS₂(=ruthenium(II)tris-(4,4′diphenyl-2,2′-bipyridyl)(3-trimethylsilyl)-1-propanesulfonate) were added to that solution. By knife application, thesolution was drawn up a polyester foil (Mylar, Goodfellow, UK) with athickness of 125 μm when exhibiting a wet thickness of 125 μm. Afterevaporation of the solvent, an approximately 10 μm thick, CO2 sensitivelayer was obtained.

[0142] 3.2 Rules for Manufacturing the CO2 Sensitive Layer of an OpticalCO2 Sensor

[0143] 5 g ethylcellulose (Aldrich) were dissolved in toluol/ethanol(20/80, v/v). 100 μL tetraoctyl ammonium hydroxide (611 mmol/kgethylcellulose) and 1.08 mg cresol purple-TDMA (24 mmol/kgethylcellulose) were added to 1 g of this solution. 700 μL (containing10.1 mg dry weight of particles) of the ethanolic particle suspension(preparation by analogy with 1.2, however, by means of the dyeRu(diph-bpy)₃TMS₂) were added to that solution. By knife application,the suspension was drawn up a polyester foil (Mylar, Goodfellow, UK)with a thickness of 125 μm when exhibiting a wet thickness of 125 μm.After evaporation of the solvent, an approximately 10 μm thick, CO2sensitive layer was obtained.

[0144] The measuring results are shown in FIGS. 3 and 4 and in Table 4,respectively. The results dicate that a CO2 sensor of the inventionbased upon the FRET-principle exhibits a significantly lower O2sensitivity than a sensor produced in accordance with the state of theart. TABLE 4 Comparison of the two sensors of FIG. 1 (C1) and FIG. 2(C2). The error indications (two columns on the right) clearly showthat, with the sensor according to the invention, the O2 error issignificantly smaller. C1 C2 indicated decay time (μs) decay time (μs)pCO₂ error oxygen- oxygen- air in hPa pCO₂ (hPa) free air saturated freesaturated C1 C2 0 1.05 0.74 2.19 2.14 +7 +1 30 2.17 1.44 3.29 3.12 >800+12 60 2.41 1.56 3.52 3.36 >800 +15 100 2.71 1.70 3.81 3.61 >800 +46

Example 4 (CO2 sensor)

[0145] This sensor consists of a nanoporous membrane (Teflon membrane ofMillipore) coated with a thin film of polyacryl nitrile into which thedonor dye is embedded. This is done by simple soaking of the porousmembrane with the donor cocktail and subsequent evaporation of thesolvent. Subsequently, a second coating is prepared by analyte-sensitivecolour chemistry (in that case, a CO₂ sensor). Thus, donor and acceptorare provided in separate phases.

[0146] Manufactoring Rules:

[0147] A nanoporous Teflon filter is soaked with a 5% polyacryl nitrileruthenium complex solution. Subsequently, the solvent is left toevaporate (100° C. for 10 hours). In a second step, the same porousmembrane is soaked with the CO2 sensitive cocktail. The cocktail has thefollowing composition: Ethylcellulose: 1 g Ethanol: 4 ml Toluene: 16 mlTetraoctyl ammonium hydroxide: 250 mg m-cresol purple-TDMA: 30 mg

[0148] Afterwards, the filter is dried and the sensor is ready for use.30% decline of the decay time between 100% CO2 and 0% CO2.

1. An optochemical sensor functioning in accordance with theFRET-principle and exhibiting an acceptor (chromophore or luminophore)responsive to an analyte contained in a sample medium as well as a donor(luminophore), characterized in that acceptor and donor are located inseparate chemical phases, whereby the phase containing the donor isessentially impermeable to the sample medium or to components of thesample medium affecting the luminescence characteristics of theluminophore.
 2. A sensor according to claim 1, characterized in thatdonor and acceptor are each present in a matrix material, whereby thematrix material of the acceptor and donor phases is provided in mixedform or in the form of a thin layer (sensor layer), respectively.
 3. Asensor according to claim 2, characterized in that the sensor layer isattached to a transparent substrate or to a light guide.
 4. A sensoraccording to claim 2 or 3, characterized in that an optical insulationlayer permeable to the analyte and located between the sensor layer andthe substance to be analyzed is provided.
 5. A sensor according to anyof claims 2 to 4, characterized in that the sensor layer consists of athin porous material, the pores of that material containing the matrixmaterial of the donor phase and the matrix material of the acceptorphase (FIG. 5).
 6. A sensor according to any of claims 2 to 4,characterized in that the sensor layer is composed of a thin film, saidthin film representing the matrix material of the acceptor phase (FIG.6).
 7. A sensor according to claim 6, characterized in that the matrixmaterial of the donor phase is provided in homogeneously distributedfashion in the matrix material of the acceptor phase.
 8. A sensoraccording to any of claims 2 to 7, characterized in that the donor phaseis provided in the form of particles.
 9. A sensor according to claim 8,characterized in that the acceptor molecules are attached to the surfaceof the particles of the donor phase.
 10. A sensor according to any ofthe preceding claims, characterized in that unplasticized polymers areused as a material for the donor phase.
 11. A sensor according to claim10, characterized in that polyacryl nitrile and derivatives thereof, PVCand/or polyvinylidene chloride are used as polymers.
 12. A sensoraccording to any of the preceding claims, characterized in thatpolyvinyl chloride, polystyrenes, polycarbonates, polyethylenes,polyurethanes, silicones, copolymers of polyvinyl alcohol and polyvinylacetate, copolymers of polyvinyl chloride, polyvinyl alcohol andpolyvinyl acetate, cellulose, polyurethanes with hydrophilic groups,polyhydroxyethylmethacrylates, crosslinked polyvinyl alcohols, and/orpolyacrylamides, optionally with up to 80% of plasticizer, are used asmaterials for the acceptor phase.
 13. A method for qualitative and/orquantitative determination of at least one analyte and/or component of agaseous or liquid measuring medium according to the FRET-principle,characterized by the use of a sensor according to any of claims 1 to 12.14. A method according to claim 13, characterized in that it is carriedout for the determination of the pH-value of a sample, for thedetermination of concentrations and/or activities of ions in a sample orfor the determination of components exhibiting acid or alkalinereactions in aqueous media while being gaseous under normal conditions.15. A method according to claim 14, characterized in that it is carriedout for the determination of concentrations and/or activities of Li⁺,Na⁺, K⁺, Mg⁺⁺, Ca⁺⁺ or Cl⁻.
 16. A method according to claim 14,characterized in that it is carried out for the determination of CO₂ orNH₃ in a liquid measuring medium.
 17. A method according to any ofclaims 13 to 16, characterized in that the measuring medium is a bodyfluid, in particular blood, plasma or serum.
 18. A method according toany of claims 13 to 17, characterized in that the sensor is used as atransducer.
 19. A method according to claim 18, characterized in that anenzymatical sensor is used.